www.rsc.org/loc | Lab on a Chip
PAPER
Simultaneous particle counting and detecting on a chip Xudong Wu,a Chan Hee Chon,b Yao-Nan Wang,c Yuejun Kangb and Dongqing Li*b Received 13th March 2008, Accepted 15th July 2008 First published as an Advance Article on the web 10th September 2008 DOI: 10.1039/b804319a This paper reports a lab-on-a-chip device that performs particle detection and number counting by coupling the fluorescent detection and particle counting simultaneously. The particle number counting is realized by a resistive pulse sensor (RPS) and fluorescent particle detection is achieved by a miniaturized laser-fiber optic detection system. By using a single microfluidic channel with two detecting arm channels placed at the two ends of the sensing section, the RPS signal-to-noise ratio is improved significantly. Two-stage differential amplification is used to further increase the signal-to-noise ratio for both the RPS and fluorescent signals. This method is also highly sensitive, so that we were able to realize the RPS and fluorescent detection of 0.9 mm (mean diameter) fluorescent particles. Excellent agreement was achieved by comparing the results obtained by our system with the results from a commercial flow cytometer for a variety of samples of mixed fluorescent and non-fluorescent particles. The method described in this paper is simple and can be applied to develop a compact device without the need of lock-in amplifier or similar bulky supplemental equipment.
Introduction The interests for counting and detecting small particles have increased in medicine, chemistry, biology, and particle science areas over the past decades. To develop low-cost and portable devices with higher throughput, sensitivity and accuracy, research efforts are focused on particle detection and analysis on an integrated lab-on-a-chip.1–3 As demonstrated in these studies, the resistive pulse sensor (RPS) and fluorescence techniques have promising advantages because of their sensitivity and simplicity.4,5 The RPS is a powerful method to detect the size of individual particles or cells and count their numbers.6–8 The RPS detects the impedance change across a small sensing channel when a non-conducting particle passes this sensing channel.9 The widely used Coulter counter exemplifies the vitality of this technology. The development of micro/nano-scale technology requires a miniaturized RPS counter to detect micro- and nanoscale particles in lab-on-a-chip devices.10–12 To detect and analyze submicron biopolymers, such as DNA, proteins, or even big molecules by artificial nano-scale pores, many researchers have reported forming a small sensing channel by using ion beam etching, carbon nanotubes or bio-layers.13–18 However, these approaches make the practical applications of nano-scale RPS difficult because of the associated disadvantages, such as high fabrication cost, instability leading to low repeatability, and high time consumption. To overcome these confronted problems, a Department of Biomedical Engineering, Chongqing University, Chongqing, 400044, China b Department of Mechanical Engineering, Vanderbilt University, Nashville, TN, 37235, USA. E-mail:
[email protected]; Fax: +1-615-3436687 c Department of Mechanical Engineering, National Cheng-Kung University, Tainan, 701, Taiwan
This journal is © The Royal Society of Chemistry 2008
many researchers work on improving RPS detection sensitivity by new designs of the microchannels and employing highlysensitive detecting methods.19,20 Recently the authors reported the detection of nano-particles in a microfluidic chip21,22 and the detection of bio-particles, such as CD4+ T lymphocytes, based on the MOSFET and fluorescent detection techniques.23 Fluorescent detection is widely applied in the microfluidicbased studies of analytical biology and chemistry24–28 due to its simplicity and high sensitivity. However, because of the weak original fluorescent signal, conventional detection relies on the lock-in amplification method including a function generator in order to achieve reasonable sensitivity. With the support of these sophisticated and bulky instruments, fluorescent detection method has been employed to detect and identify various biological or chemical samples from cells to single molecules.26–28 However, this method has a critical disadvantage for lab-on-achip applications because of its dependence on the expensive and bulky instruments. This paper reports particle counting and detection on a microfluidic chip by using a simpler and highly sensitive RPS counter and a miniaturized fluorescent detector. The system is able to detect and count non-fluorescent as well as fluorescent particles or cells simultaneously. In this system, we developed a single microchannel with two detecting arm channels (Fig. 1) to detect the resistive pulses caused by the particles. The signals were amplified by a two-stage differential amplifier which improves the signal-to-noise ratio (S/N) significantly. As a result we achieved detection of 0.9 mm (in diameter) particles using a relatively large sensing gate of 16 ¥ 50 ¥ 20 mm3 (Fig. 1). The relatively large sensing gate can be easily manufactured quickly by using the soft lithography method. By adopting two-stage differential amplification and a custom-made electronic filter system, our fluorescent detection system can also detect submicron size particles. Compared with the conventional system Lab Chip, 2008, 8, 1943–1949 | 1943
DV downstream =
R 3 DR (V + - V - ) (3) ( R1 + R 2 + R 3 ) 2 + ( R1 + R 2 + R 3 ) DR
where R1 , R2 , R3 are the resistances of the upstream section, the sensing gate and the downstream section, respectively. (V + - V - ) is the voltage drop across the main channel, and the voltage is measured at two reservoirs. Output resistive signals of two detecting arm channels were connected to the differential amplifiers. When a particle passes the sensing gate, the theoretical output voltage difference between the two detecting arm channels, which is amplified by the differential amplifier of gain A, can be written as:
Fig. 1 A schematic diagram of the system structure. In the center of the PDMS microchip, the shaded constriction area is the sensing gate. The two detecting arm channels are located next to the sensing gate at both ends. The direction of the electrokinetic flow is from V + to V - .
that has to use a lock-in amplifier, this system features high sensitivity, low cost, compact size and high accuracy.
Materials and methods This system consists of a single-gate RPS counter, a miniaturized laser-fiber optic fluorescent detector, two sets of two-stage differential amplifiers, a microfluidic chip and a LabViewR module, as shown in Fig. 1. The data acquisition module collects signals at a sampling rate of 1000 Hz and processes the results using a custom-made LabviewR code. The RPS counter detects the total numbers of fluorescent and non-fluorescent particles, and the laser-fiber optic fluorescent detector enumerates only the fluorescent particles. Single gate differential counter In this system (Fig. 1), we developed a single gate differential counter with high sensitivity based on the RPS method. By designing a single sensing gate with two detecting arm channels located at both ends of the gate (Fig. 1) and using the two-stage differential amplification, the sensitivity of RPS detection has been improved dramatically, as explained below. Theoretically, when a non-conducting particle passes through a small sensing gate, the resistance of the sensing gate is changed and the resistance change, DR, is given by29
DR =
˘ 4 rd 3 È 2d 3 2d 3 + ( 2 ) 2 + …˙ 1+ 4 Í 2 p D Î 3D L 3D L ˚
(d
